Camptothecin analogs having an E-ring ketone

ABSTRACT

Camptothecin analogs having an E-ring ketone are effective anti tumor compounds. These compounds inhibit the enzyme topoisomerase I and may alkylate DNA of the associated topoisomerase I DNA cleavable complex.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to camptothecin analogs having an E-ring ketonewhich inhibit the enzyme topoisomerase I and have anticancer activity.This invention is also related to the treatment of tumors in animalswith camptothecin analogs.

2. Background of the Invention

Camptothecin (CPT) is a naturally occurring cytotoxic alkaloid which isknown to inhibit the enzyme topoisomerase I and is a potent anti tumoragent. Camptothecin compounds have the general ring structure shownbelow.

Camptothecin was isolated from the wood and bark of Camptothecaacuminata by Wall et al. (Wall et al., 1966, J. Am. Chem. Soc., 88:3888). It has been shown that if the E-ring α-hydroxy lactone functionsare altered or removed, that the resulting compounds have no activityregarding topoisomerase I inhibition or inhibition of cancer cells.(Wall, Plant Antitumor Agents. V. Alkaloids with Antitumor ActivitySymposiumsberichtes, pp. 77 87, 4. Internationales Symposium, Biochemieund Physiologie der Alkaloide, Halle (Saale) 25. Bis 28. Jun., 1969,edited by K. Mothes, K. Schreiber, and H. R. Schutte, Akademie Verlag,Berlin, 1969; and Nicholas et al, J. Med. Chem., 33, 972 (1990).)

Another process that affects all camptothecin compounds is that at analkaline pH, as low as 7.5 or higher, the lacton E-ring is readilyhydrolyzed to give an E-ring opened carboxylate product. This compoundis much less active in the above mentioned activities.

The cytotoxic activity of camptothecin compounds is believed to arisefrom the ability of these compounds to inhibit both DNA and RNAsynthesis and to cause reversible fragmentation of DNA in mammaliancells. Topoisomerase I relaxes both positively and negativelysupercoiled DNA and has been implicated in various DNA transactions suchas replication, transcription and recombination. The enzyme mechanism isbelieved to involve a transient breakage of one of the two DNA strandsand the formation of a reversible covalent topoisomerase I enzyme DNAcomplex. Camptothecin interferes with the DNA breakage reunion reactionby reversibly trapping the enzyme DNA intermediate termed the “cleavablecomplex”. The cleavable complex assay is a standard test for determiningthe cytotoxic activity of camptothecin compounds. The high levels oftopoisomerase I in several types of human cancer and the low levels incorrespondingly normal tissue provide the basis for tumor treatment withbiologically active camptothecin analogs.

U.S. Pat. No. 4,894,456 describes methods of synthesizing camptothecincompounds which act as inhibitors of topoisomerase I and are effectivein the treatment of leukemia (L 1210). U.S. Pat. No. 5,225,404 disclosesmethods of treating colon tumors with camptothecin compounds.

Numerous camptothecin compounds and their use as inhibitors oftopoisomerase I are reported by U.S. Pat. No. 5,053,512; U.S. Pat. No.4,981,968; U.S. Pat. No. 5,049,668; U.S. Pat. No. 5,106,742; U.S. Pat.No. 5,180,722; U.S. Pat. No. 5,244,903; U.S. Pat. No. 5,227,380; U.S.Pat. No. 5,122,606; U.S. Pat. No. 5,122,526; and U.S. Pat. No.5,340,817.

U.S. Pat. No. 4,943,579 discloses the esterification of the hydroxylgroup at the 20 position of camptothecin to form several prodrugs. Thispatent further discloses that the prodrugs are water soluble and areconverted into the parent camptothecin compounds by hydrolysis.

Brangi et al., Cancer Research, 59, 5938 5946 Dec. 1, 1999, reports aninvestigation of Camptothecin resistance in cancer cells and reports thecompound difluoro 10, 11 methylenedioxy 20(S) camptothecin.

A need continues to exist, however, for camptothecin analogs havingimproved stability under physiological conditions.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide acamptothecin analog having improved stability under physiologicalconditions.

Another object of the present invention is to provide a method oftreating leukemia or solid tumors in a mammal in need thereof byadministration of a camptothecin analogs.

Another object of the present invention is to provide a method ofinhibiting the enzyme topoisomerase I and/or alkylating DNA ofassociated DNA topoisomerase I by contacting a DNA topoisomerase Icomplex with a camptothecin analog.

These and other objects of the present invention are made possible by acamptothecin analog having the structure:

-   -   where    -   X and Y are each independently NO₂, NH₂, H, F, Cl, Br, I, COOH,        OH, O—C₁₋₆ alkyl, SH, S—C₁₋₆ alkyl, CN, NH—C₁₋₆ alkyl, N(C₁₋₆        alkyl)₂, CHO, C₁₋₈ alkyl, N₃,    -   -Z-(CH₂)_(a)—N—((CH₂)_(b)OH)₂, wherein Z is selected from the        group consisting of O, NH and S, and a and b are each        independently an integer of 2 or 3,    -   -Z-(CH₂)₂—N—(C₁₋₆ alkyl)₂ wherein Z is selected from the group        consisting of O, NH and S, and a is an integer of 2 or 3,    -   —CH₂-L, where L is halogen (F, Cl, Br, I), ⁺N₂, ⁺(OR¹)₂,        ⁺S(R¹)₂, ⁺N(R¹)₃, OC(O)R¹, OSO₂R¹, OSO₂CF₃, OSO₂C₄F₉, C₁₋₆        alkyl-C(═O)—, C₄₋₁₈ aryl-C(═O)—, C-₁₋₆ alkyl SO-2-, perfluoro        C₁₋₆alkyl-SO₂— or C₄₋₁₈ aryl-SO₂—, (where each R¹ independently        is C₁₋₆ alkyl, C₄₋₁₈ aryl or C₄₋₁₈ArC₁₋₆ alkyl); or —CH₂NR²R³,        where (a) R² and R³ are, independently, hydrogen, C₁₋₆ alkyl,        C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl,        hydroxyl-C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ COR⁴ where R⁴ is hydrogen,        C₁₋₆ alkyl, perhalo-C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇        cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl, hydroxyl-C₁₋₆ alkyl, C₁₋₆        alkoxy, or C₁₋₆ alkoxy-C₁₋₆ alkyl, or (b) R² and R³ taken        together with the nitrogen atom to which they are attached form        a saturated 3-7 membered heterocyclic ring which may contain a        O, S or NR⁵ group, where R⁵ is hydrogen, C₁₋₆ alkyl,        perhalo-C₁₋₆ alkyl, aryl, aryl substituted with one or more        groups selected from the group consisting of C₁₋₆ alkyl,        halogen, nitro, amino, C₁₋₆ alkylamino, perhalo-C₁₋₆ alkyl,        hydroxyl-C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl and        —COR⁶ where R⁶ is hydrogen, C₁₋₆ alkyl perhalo-C₁₋₆ alkyl, C₁₋₆        alkoxy, aryl, and aryl substituted with one or more C₁₋₆ alkyl,        perhalo-C₁₋₆ alkyl, hydroxyl-C₁₋₆ alkyl, or C₁₋₆ alkoxy-C₁₋₆        alkyl groups;    -   R⁷ is H, or C(O)—(CH₂)_(m)—NR⁸R⁹, where m is an integer of 1-6        or —C(O)CHR¹⁰NR⁸R⁹, where R¹⁰ is the side chain of one of the        naturally occurring α-amino acids, R⁸ and R⁹ are, independently,        hydrogen, C₁₋₈ alkyl or —C(O)CHR¹¹NR¹² R³, where R¹¹ is the side        chain of one of the naturally occurring α-amino acids and R¹²        and R¹³ are each independently hydrogen or C₁₋₈ alkyl;    -   W is independently H or F,    -   R¹³ and R¹⁴ are each H or combine to form a double bond; and    -   n is an integer of 1 or 2,    -   and salts thereof.

These compounds have the necessary α-hydroxy-ethyl substitutents at C₂₀and a ketone in place of the lactone structure. Such a compound has aspatial orientation virtually identical with that of camptothecin,however it is much more stable than CPT under alkaline conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same become betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 illustrates the synthesis of a tricyclic ketone used to form thecompound of the present invention;

FIG. 2 illustrates a synthetic reaction scheme for preparing compoundsaccording to the present invention; and

FIG. 3 illustrates a synthetic reaction scheme for preparing compoundsaccording to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless indicated to the contrary, the term “alkyl” as used herein meansa straight chain or branched chain alkyl group with 1-30, preferably1-18 carbon atoms, more preferably 1-8 carbon atoms, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl,n-decyl, undecyl, dodecyl, myristyl, heptadecyl and octadecyl groups.The term “alkyl” also includes C₃₋₃₀ cycloalkyl groups such ascyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl groups.

Unless indicated to the contrary, the term “aryl” as used herein means acarbocyclic aromatic ring having 6-18 carbon atoms, preferably 6 10carbon atoms in the aromatic ring structure. The aromatic rings may besubstituted by one or more alkyl group, preferably alkyl groups having1-10 carbon atoms. A particularly preferred aryl group is phenyl.

Unless indicated to the contrary, the term “aralkyl” as used hereinmeans a straight chain or branched chain alkyl group as defined abovefor the term “alkyl” bonded to an aryl group as defined above for theterm “aryl”. Preferred aralkyl groups are benzyl, phenethyl, etc.

As used herein, the term “acyl” means formyloxy and acyl moietiesderived from aromatic carboxylic acids, heterocyclic carboxylic acids,aralkyl carboxylic acids, as well as alkyl and aromatic sulfonic acids.The alkyl groups of these acyloxy moieties may be a straight chain orbranched chain alkyl group with 1-7 carbon atoms. Additionally, the acylmoiety may contain one or more unsaturated carbon carbon bonds and mayalso carry one or more substituents such as halogen, amino and hydroxylgroups.

The camptothecin analogs of the present invention may bear a leavinggroup at one or more of the positions C₇ or Cg of the camptothecin ringstructure. More specifically, the leaving group is a group of theformula —CH₂-L, where L is a functional group which can be easilydisplaced, i.e. L is a good leaving group in nucleophilic substitutionreactions. Suitable groups L include halogen (F, Cl, Br, I), ⁺N₂,⁺O(R¹)₂, ⁺S(R¹)₂, ⁺N(R¹)₃, OC(O)R¹, OSO₂R¹, OSO₂CF₃, and OSO₂C₄F₉,C₁₋₆alkyl-C(═O)—, C₄₋₁₈aryl-C(═O)—, C₁₋₆ alkyl-SO₂—, perfluoroC₁₋₆alkyl-SO₂— and C₄₋₁₈ aryl-SO₂—, (where each R¹ independently is C₁₋₆alkyl, C₄₋₁₈ aryl or C₄₋₁₈ ArC₁₋₆ alkyl).

While not being bound by any particular theory, it is believed thatnucleophilic groups on DNA displace leaving group L from thecamptothecin analogs of the present invention resulting in alkylation ofthe DNA by the alkylating group of the camptothecin ring structure.Suitable nucleophilic groups present in DNA include the nucleophilicgroups found in DNA bases adenine, guanine, thymine, and cytosine, suchas NH₂, —NH— and ═N— groups. When a camptothecin analog of the inventionhaving a —CH₂-L group is contacted with DNA, nucleophilic displacementof leaving group L results in alkylation of the nucleic acid. Thecompounds of the present invention exhibit a novel anti tumor activityby alkylating DNA.

Camptothecin analogs have an asymmetric carbon atom at the 20-positionmaking two enantiomeric forms, i.e., the (R) and the (S) configurations,possible. This invention includes each enantiomeric form individually,as well as combinations or mixtures of these forms. The invention alsoincludes other forms of the camptothecin analogs including solvates,hydrates, polymorphs, salts, etc. Particularly preferred compounds arecamptothecin derivatives having the (S) configuration at the20-position.

In a preferred embodiment, X is NO₂, NH₂, H, F, Cl, Br, I, COOH, OH,O—C₁₋₆ alkyl, SH, S—C₁₋₆ alkyl, CN, CH₂NH₂, NH—C₁₋₆ alkyl, CH₂NH—C₁₋₆alkyl, N(C₁₋₆ alkyl)₂, CH₂N(C₁₋₆ alkyl)₂, O—CH₂CH₂N(CH₂CH₂OH)₂,NH—CH₂CH₂N(CH₂CH₂OH)₂, S—CH₂CH₂N(CH₂CH₂OH)₂, O—CH₂CH₂CH₂N(CH₂CH₂OH)₂,NH—CH₂CH₂CH₂N(CH₂CH₂OH)₂, S—CH₂CH₂CH₂N(CH₂CH₂OH)₂,O—CH₂CH₂N(CH₂CH₂CH₂OH)₂, NH—CH₂CH₂N(CH₂CH₂CH₂OH)₂,S—CH₂CH₂N(CH₂CH₂CH₂OH)₂, O—CH₂CH₂CH₂N(CH₂CH₂CH₂OH₂)₂,NH—CH₂CH₂CH₂N(CH₂CH₂CH₂OH₂)₂, S—CH₂CH₂CH₂N(CH₂CH₂CH₂OH₂)₂,O—CH₂CH₂N(C₁₋₆ alkyl)₂, NH—CH₂CH₂N(C₁₋₆ alkyl)₂, S—CH₂CH₂N(C₁₋₆ alkyl)₂,O—CH₂CH₂CH₂N(C₁₋₆ alkyl)₂, NH—CH₂CH₂CH₂N(C₁₋₆ alkyl)₂, S—CH₂CH₂CH₂N(C₁₋₆alkyl)₂, CHO, N₂, C₁₋₈ alkyl, CH₂-L where L is halogen (F, Cl, Br, I),⁺N₂, ⁺O(R¹)₂ (where each R¹ independently is alkyl, aryl or aralkyl asdefined above), ⁺S(R¹)₂, ⁺N(R¹)₃, OC(O)R¹, OSO₂R¹, OSO₂CF₃, OSO₂C₄F₉,C₁₋₆alkyl-C(═O)—, C₄₋₈aryl-C(═O)—, C₁₋₆alkyl-SO₂—, perfluoroC₁₋₆alkyl-SO₂— and C₄₋₁₈aryl-SO₂—.

In a preferred embodiment Y is H, C₁₋₈ alkyl, or CH₂NR²R³ where (a) R²and R³ are, independently, hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl, hydroxyl-C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ COR⁴ where R⁴ is hydrogen, C₁₋₆ alkyl, perhalo C₁-alkyl,C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl, hydroxyl-C₁₋₆alkyl, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl, or (b) R² and R³ takentogether with the nitrogen atom to which they are attached form asaturated 3-7 membered heterocyclic ring which may contain a O, S or NR⁵group, where R⁵ is hydrogen, C₁₋₆ alkyl, perhalo C₁₋₆ alkyl, aryl, arylsubstituted with one or more groups selected from the group consistingof C₁₋₆ alkyl, halogen, nitro, amino, C-6 alkylamino, perhalo-C₁₋₆alkyl, hydroxyl-C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆-alkoxy-C₁₋₆ alkyl and—COR⁶ where R⁶ is hydrogen, C₁₋₆ alkyl perhalo-C₁₋₆ alkyl, C₁₋₆ alkoxy,aryl, and aryl substituted with one or more C₁₋₆ alkyl, perhalo-C₁₋₆alkyl, hydroxyl-C₁₋₆ alkyl, or C₁₋₆ alkoxy-C₁₋₆ alkyl groups.

The group R⁷ may be an ester of a naturally occurring or non naturallyoccurring amino acid such as an ester of glycine or β-alanine. Inparticular, the present invention is directed to camptothecin analogswhere the group R⁷ is C(O)—(CH₂)_(m)—NR⁸R⁹, where m is the integer 1, 2,3, 4, 5 and 6 and R⁸ and R⁹ are each H.

Suitable side chains R¹⁰ and R¹ appearing on the group R⁷ are the sidechains of the amino acids glycine, α-alanine, β-alanine, valine,leucine, isoleucine, phenylalanine, tyrosine, tryptophan, lysine,arginine, histidine, aspartate, glutamate, asparagine, glutamine,cysteine and methionine. Moreover, the group R⁷, may comprise two aminoacid units linked by a peptide linkage. In particular the group R⁷ maycomprise a β-alanine group linked to a lysine of the structure

Moreover, the group R⁷ may provide basis for the formation of a mono ordi salts, via the free amine groups, such as a hydrochloride ordihydrochloride.

A synthon for attaching such a group to a terminal hydroxyl group isdescribed by Hudkins et al. Bioorg. Med. Chem. Lett, 8 (1998) 18731876).

Particularly preferred esters are glycinate esters and the peptide esterbased on β-alanine lysine. These esters are pro drugs which areconverted to the camptothecin analog compound by hydrolysis of the esterbond. The esters may be prepared by the method described in U.S. Pat.No. 4,943,579 which is incorporated herein by reference for a morecomplete description of the process of preparing the esters and for adescription of suitable esters formed by the process. The esterificationsynthon may need to introduced in a protected form, such that thereaction of amine groups is inhibited, followed by removal of theprotecting group. Such protecting groups are well known to those ofordinary skill in the art and are described by Hudkins et al. Bioorg.Med. Chem. Lett, 8 (1998) 1873 1876).

Specific examples of non limiting compounds include10,11-difluoromethylenedioxy-20-(S)-camptothecin E-ring ketone; 7-ethyl10,11-difluoromethylenedioxy-20-(S)-camptothecin E-ring ketone;7-chloromethyl 10,11-difluoromethylenedioxy-20-(S)-camptothecin E-ringketone; 7-bromomethyl 10,11-difluoromethylenedioxy-20-(S)-camptothecinE-ring ketone; 7-hydroxymethyl10,11-difluromethylenedioxy-20-(S)-camptothecin E-ring ketone, 9-nitro10,11-difluoromethylenedioxy-20-(S)-camptothecin E-ring ketone, 9-amino10,11-difluoromethylenedioxy-20-(S)-camptothecin E-ring ketone,7-ethyl-9-nitro 10,11-difluoromethylenedioxy-20-(S)-camptothecin E-ringketone and 7-ethyl-9-amino10,11-difluoromethylenedioxy-20-(S)-camptothecin E-ring ketone.

Specific non limiting examples further include the C₂₀ amino acid esterof the above identified compounds10,11-difluoromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone; 7-ethyl10,11-difluoromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone; 7-chloromethyl10,11-difluoromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone; 7-bromomethyl10,11-difluoromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone; 7-hydroxymethyl10,11-difluromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone, 9-nitro10,11-difluoromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone, 9-amino10,11-difluoromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone, 7-ethyl-9-nitro10,11-difluoromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone, 7-ethyl-9-amino10,11-difluoromethylenedioxy-20-O-glycinyl-20-(S)-camptothecin E-ringketone,10,11-difluoromethylenedioxy-20-O—N-methylglycinyl-20-(S)-camptothecinE-ring ketone; 7-ethyl 10,11-difluoromethylenedioxy 20-O—Nmethylglycinyl-20-(S)-camptothecin E-ring ketone; 7-chloromethyl10,11-difluoromethylenedioxy 20-O—N methylglycinyl-20-(S)-camptothecinE-ring ketone; 7-bromomethyl 10,11-difluoromethylenedioxy 20-O—Nmethylglycinyl-20-(S)-camptothecin E-ring ketone; 7-hydroxymethyl 10, 11difluromethylenedioxy 20-O—N methylglycinyl-20-(S)-camptothecin E-ringketone, 9-nitro 10,11-difluoromethylenedioxy 20-O—Nmethylglycinyl-20-(S)-camptothecin E-ring ketone, 9-amino10,11-difluoromethylenedioxy 20-O—N methylglycinyl-20-(S)-camptothecinE-ring ketone, 7-ethyl-9-nitro 10,11-difluoromethylenedioxy 20-O—Nmethylglycinyl-20-(S)-camptothecin E-ring ketone, 7-ethyl-9-amino10,11-difluoromethylenedioxy 20-O—N methylglycinyl-20-(S)-camptothecinE-ring ketone, 10,11-difluoromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone; 7-ethyl10,11-difluoromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone; 7-chloromethyl10,11-difluoromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone; 7-bromomethyl10,11-difluoromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone; 7-hydroxymethyl10,11-difluromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone, 9-nitro10,11-difluoromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone, 9-amino10,11-difluoromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone, 7-ethyl-9-nitro10,11-difluoromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone and 7-ethyl-9-amino10,11-difluoromethylenedioxy 20-O—N,Ndimethylglycinyl-20-(S)-camptothecin E-ring ketone.

Additional specific non limiting examples further include10,11-difluoromethylenedioxy-20-O—O-ala-lys-20-(S)-camptothecin E-ringketone; 7-ethyl10,11-difluoromethylenedioxy-20-O—O-ala-lys-20-(S)-camptothecin E-ringketone; 7-chloromethyl10,11-difluoromethylenedioxy-20-O-β-ala-lys-20-(S)-camptothecin E-ringketone; 7-bromomethyl10,11-difluoromethylenedioxy-20-O-β-ala-lys-20-(S)-camptothecin E-ringketone; 7-hydroxymethyl10,11-difluromethylenedioxy-20-O-β-ala-lys-20-(S)-camptothecin E-ringketone, 9-nitro10,11-difluoromethylenedioxy-20-O-β-ala-lys-20-(S)-camptothecin E-ringketone, 9-amino10,11-difluoromethylenedioxy-20-O-β-ala-lys-20-(S)-camptothecin E-ringketone, 7-ethyl-9-nitro10,11-difluoromethylenedioxy-20-O-β-ala-lys-20-(S)-camptothecin E-ringketone and 7-ethyl-9-amino10,11-difluoromethylenedioxy-20-O-β-ala-lys-20-(S)-camptothecin E-ringketone.

Additional specific non limiting examples further include10,11-difluoromethylenedioxy-20-O-β-ala-20-(S)-camptothecin E-ringketone; 7-ethyl10,11-difluoromethylenedioxy-20-O-B-ala-20-(S)-camptothecin E-ringketone; 7-chloromethyl10,11-difluoromethylenedioxy-20-O-β-ala-20-(S)-camptothecin E-ringketone; 7-bromomethyl10,11-difluoromethylenedioxy-20-O-β-ala-20-(S)-camptothecin E-ringketone; 7-hydroxymethyl10,11-difluromethylenedioxy-20-O-β-ala-20-(S)-camptothecin E-ringketone, 9-nitro10,11-difluoromethylenedioxy-20-O-β-ala-20-(S)-camptothecin E-ringketone, 9-amino10,11-difluoromethylenedioxy-20-O-β-ala-20-(S)-camptothecin E-ringketone, 7-ethyl-9-nitro10,11-difluoromethylenedioxy-20-O-β-ala-20-(S)-camptothecin E-ringketone and 7-ethyl-9-amino10,11-difluoromethylenedioxy-20-O-β-ala-20-(S)-camptothecin E-ringketone.

The compounds of the present invention may be prepared by conventionalmethods known to those of ordinary skill in the art, without undueexperimentation.

For example, the claimed compounds may be prepared by condensation of aaminophenylcarbonyl of formula IV or V

-   -   where X, Y, W and n are as defined for formula I    -   with a tricyclic ketone of formula III    -   where R¹³ and R¹⁴ are defined as for formula I.

The condensation reaction is analogous to the condensation reactiondescribed by Wall et al. U.S. Pat. No. 5,122,526, the relevant portionsof which are hereby incorporated by reference.

The synthetic sequence is described with reference to FIG. 1. The20-desoxy tricyclic analog (1) is treated with an appropriate amine asshown in the example. It is a cyclopentyl amine and the correspondingamide (2) is obtained. On acetylation, 2 is converted to the acetate 3.The next step is a homologolation reaction. Other homologolationreactions may be carried out by converting 2 to a bromide, etc. Thecompound 4 is converted to a hydroxy derivative containing one morecarbon atom, compound 5. Compound 5 may be brominated to the bromoanalog 6. Compound 6 in turn is converted as shown to the tricyclic20-desoxy ketone 7. Compound 7 may be hydroxylated to the(RS)-20-hydroxy compound 8. Finally, acidic cleavage of the ketal yieldsthe 20(RS)-hydroxy ketone 9 which is the reactant that can be convertednow to many camptothecin analogs (as shown in the attached example).

Additional, non limiting examples of condensation reactions areillustrated in FIG. 2.

Alternatively, suitable E-ring ketone compounds may also be preparedfrom the corresponding compothecin compound bearing an E-ring lactone,illustrated in FIG. 3, by the following reaction sequence:

-   -   i) reaction of the E-ring lactone with a primary alkylamine to        form an α-hydroxy alkylamide;    -   ii) activation of a pendant D ring hydroxymethylene group to        form a leaving group, such as by formation of an acetate,        followed by a displacement reaction with a dialkyl malonate,        such as diethylmalonate;    -   iii) esterification of the alkylamide to an alkyl ester;    -   iv) cyclization of a D-ring methylene malonate onto the alkyl        ester to form the E-ring ketone followed by decarboxylation of        an ester group; and    -   v) decarboxylation of the remaining ester group.

In a further embodiment, an α-β E-ring unsaturation may be introduced byconventional methods known to those of ordinary skill in the art, suchas by reacion with DDQ.

An alternative procedure for preparing the E-ring ketone fromcamptothecin is attached and involves a procedure as shown. If theE-ring ketone can be prepared at the very end, a conjugated B ring canbe prepared as shown in the very last step. Such a compound might havevery interesting properties. It might possibly intercalate with DNAwhereas camptothecin does not.

Substitution at the C₇ position may be conducted by condensation withthe corresponding aldehyde of the C₇ substituent. Esterification with anamino acid at C₂₀ is possible by conventional methods known to those ofordinary skill in the art. Substitution at C₉ with groups such a nitroand amino is also possible in a manner analogous to that described inthe literature.

The compounds of the invention having the group —CH₂-L at C₉ areprepared from known 20(S)—CPT compounds bearing a halogen, for example,a bromine atom, at the C₉ position. The halogen atom can be readilyconverted into the corresponding cyano analog by reaction with CuCN,followed by hydrolysis to form the corresponding carboxy analog. Thecarboxy analog is reduced to the corresponding hydroxy methyl analogwhich can be reacted with Ph₃P—CCl₄ to provide the correspondingchloromethyl analog. The chloromethyl analog can be readily converted tothe bromomethyl and iodomethyl analogs using LiBr or LiI. The remainingcompounds of the invention are prepared from these compounds by reactionwith the corresponding acid chloride, sulfonyl chloride, etc. Thesereactions are well known to one having ordinary skill in this art.

Compounds in which L is Br or I are readily prepared from the compoundin which L is Cl by simple halide exchange employing LiBr or LiI indimethylformamide (DMF) solution (Larock, R. C., Comprehensive OrganicTransformations, VCH Publishers, Inc., p. 337, N.Y. 1989).

Alternatively, the 7-methyl compounds (L is H) can be prepared either bya Friedlander reaction employing the corresponding acetophenone, or by afree radical alkylation reaction (Sawada et al., 1991, Chem. Pharm.Bull., 39: 2574). Free radical bromination of 7-methyl substrates can beaccomplished by employing N-bromosuccinimide (NBS) in acetic acid (HOAc)under catalysis by benzoyl peroxide to give compounds in which L is Br.

9-Nitro-difluoro-10,11-methylenedioxy-20-(S)-camptothecin may beprepared from difluoro-10,11-methylenedioxy-20-(S)-camptothecin bytreatment with HNO₃. 9-Aminodifluoro-10,11-methylenedioxy-20-(S)-camptothecin may be prepared from9-nitro difluoro-10,11-methylenedioxy-20-(S)-camptothecin via reductionwith SnCl₂.

Other compounds which possess oxygen derived leaving groups, such astriflate or tosylate, are prepared from the 7-hydroxymethyl and/or7-halomethyl compounds. The 7-hydroxymethyl compounds are prepared fromthe corresponding parent compounds by the hydroxymethylation reaction.(e.g. Sawada et al., 1991, Chem. Pharm. Bull., 39: 2574) Treatment ofthese compounds with readily available sulfonic acid chlorides oranhydrides using known procedures (Stang et al., 1982, Synthesis, 85)provides the highly electrophilic substrates noted above. Alternatively,the compounds described above can be generated from any of thesubstrates where L is Cl, Br or I by reaction with the silver salt ofthe corresponding acid (e.g., silver trifluoromethanesulfonate, silvertosylate, etc.) as described generally by Stang et al. and morespecifically by Gramstad and Haszeldine (T. Gramstad and R. N.Haszeldine, 1956, J. Chem. Soc., 173).

C₂₀ esters may be prepared by esterifying the 20-position hydroxyl groupof a camptothecin analog to form an ester containing a water solublemoiety. Generally, the camptothecin analog is initially suspended inmethylene chloride or other inert solvent, stirred and cooled. To thecooled mixture is added one equivalent of an acid having the formulaHOOC—CHR¹⁰—NR⁸R⁹ or HOOC—(CH₂)_(m)—NR⁸R⁹, where m is an integer from1-6, preferably 2-6, and R¹⁰ is the side chain of one of the naturallyoccurring α-amino acids. R⁸ and R⁹ are, independently, hydrogen or C₁₋₈alkyl. Suitable side chains R¹⁰ are the side chains of the amino acidsglycine, α-alanine, β-alanine, valine, leucine, isoleucine,phenylalanine, tyrosine, tryptophan, leucine, arginine, histidine,aspartate, glutamate, asparagine, glutamine, cysteine and methionine.Particularly preferred esters are glycinate esters. One equivalent ofdicyclohexylcarbodiimide (DCC) and a catalytic amount of an amine base,preferably a secondary or tertiary amine, are also added to the mixture,which is then stirred to complete the reaction. Any precipitate whichforms is removed by filtration and the product is isolated after removalof the solvent.

The free amine(s) may be converted to an acid addition salt by theaddition of a pharmaceutically acceptable acid. Suitable acids includeboth inorganic and organic acids. Suitable addition salts include, butare not limited to hydrochloride, sulfate, phosphate, diphosphate,hydrobromide, nitrate, acetate, malate, maleate, fumarate, tartrate,succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate,palmoate, salicylate and stearate salts. The salts may be purified bycrystallization from a suitable solvent.

The water soluble 20-hydroxyl esters of the present invention aresubstantially less toxic than the parent compounds from which the estersare prepared.

The camptothecin analogs are administered in a dose which is effectiveto inhibit the growth of tumors. As used herein, an effective amount ofthe camptothecin analog is intended to mean an amount of the compoundthat will inhibit the growth of tumors, that is, reduce the site ofgrowing tumors relative to a control in which the tumor is not treatedwith the camptothecin analog. These effective amounts are generally fromabout 1-60 mg/kg of body weight per week, preferably about 2-20 mg/kgper week.

The compounds of the present invention may be administered as apharmaceutical composition containing the camptothecin analog and apharmaceutically acceptable carrier or diluent. The active materials canalso be mixed with other active materials which do not impair thedesired action and/or supplement the desired action. The activematerials according to the present invention can be administered by anyroute, for example, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid or solid form.

For the purposes of parenteral therapeutic administration, the activeingredient may be incorporated into a solution or suspension. Thesolutions or suspensions may also include the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Another mode of administration of the compounds of this invention isoral. Oral compositions will generally include an inert diluent or anedible carrier. For the purpose of oral therapeutic administration, theaforesaid compounds may be incorporated with excipients and used in theform of tablets, gelatine capsules, troches, capsules, elixirs,suspensions, syrups, wafers, chewing gums and the like. Compositions maybe prepared according to any method known to the art for the manufactureof pharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents. Tabletscontaining the active ingredient in admixture with nontoxicpharmaceutically acceptable excipients which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate granulating anddisintegrating agents, such as maize starch, or alginic acid; bindingagents, such as starch, gelatin or acacia; and lubricating agents, suchas magnesium stearate, stearic acid or talc. Tablets may be uncoated ormay be coated by known techniques to delay disintegration and adsorptionin the gastrointestinal tract and thereby provide a sustained actionover a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, such as peanut oil, liquid paraffinor olive oil.

The tablets, pills, capsules, troches and the like may contain thefollowing ingredients: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, corn starch and thelike; a lubricant such as magnesium stearate or Sterotes; a glidant suchas colloidal silicon dioxide; and a sweetening agent such as sucrose orsaccharin or flavoring agent such as peppermint, methyl salicylate, ororange flavoring may be added. When the dosage unit form is a capsule,it may contain, in addition to material of the above type, a liquidcarrier such as a fatty oil. Other dosage unit forms may contain othervarious materials which modify the physical form of the dosage unit, forexample, as coatings. Thus tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors. Materials used inpreparing these various compositions should be pharmaceutically orveterinarially pure and non-toxic in the amounts used.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylethyl cellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono oleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame, saccharin, or sucralose.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oil suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water may beformulated from the active ingredients in admixture with a dispersing,suspending and/or wetting agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose disclosed above. Additional excipients, for example sweetening,flavoring and coloring agents, may also be present.

The pharmaceutical composition of the invention may also be in the formof oil in water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents include naturallyoccurring gums, such as gum acacia and gum tragacanth, naturallyoccurring phosphatides, such as soybean lecithin, esters or partialesters derived from fatty acids and hexitol anhydrides, such as sorbitanmono-oleate, and condensation products of these partial esters withethylene oxide, such as polyoxyethylene sorbitan mono oleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent,such as a solution of 1,3-butanediol. Among the acceptable vehicles andsolvents that may be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils mayconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono ordiglycerides. In addition, fatty acids such as oleic acid may likewisebe used in the preparation of injectables. Sterilization may beperformed by conventional methods known to those of ordinary skill inthe art such as by aseptic filtration, irradiation or terminalsterilization (e.g. autoclaving).

Aqueous formulations (i.e oil in water emulsions, syrups, elixers andinjectable preparations) may be formulated to achieve the pH of optimumstability. The determination of the optimum pH may be performed byconventional methods known to those of ordinary skill in the art.Suitable buffers may also be used to maintain the pH of the formulation.

The compounds of this invention may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable nonirritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperatures and will therefore melt in the rectum to release thedrug. Non limiting examples of such materials are cocoa butter andpolyethylene glycols.

They may also be administered by intranasal, intraocular, intravaginal,and intrarectal routes including suppositories, insufflation, powdersand aerosol formulations.

The compounds of the present invention may also be administered in theform of liposome or microvesicle preparations. Liposomes aremicrovesicles which encapsulate a liquid within lipid or polymericmembranes. Liposomes and methods of preparing liposomes are known andare described, for example, in U.S. Pat. No. 4,452,747, U.S. Pat. No.4,448,765, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,721,612, U.S. Pat.No. 4,594,241, U.S. Pat. No. 4,302,459 and U.S. Pat. No. 4,186,183. Thedisclosures of these U.S. patents are incorporated herein by reference.Suitable liposome preparations for use in the present invention are alsodescribed in WO-9318749-A1, J-02056431-A and EP-276783-A.

The camptothecin analogs may be used individually to inhibit the growthof tumors. Alternatively, combinations of two or more camptothecinanalogs may be used or combinations of one or more camptothecin analogswith one or more known anti tumor compounds. When a camptothecin analogis combined with a conventional anti tumor compound, the camptothecinanalog will generally be present in an amount ranging from about 1-99wt. %, preferably, 5-95 wt. % of the combined amount of camptothecin andconventional anti tumor compound. The pharmaceutical compositions notedabove may contain these combinations of compounds together with anacceptable carrier or diluent.

The ester compounds of the invention may be administered to treatleukemia and solid tumors in mammals, including humans. The esters ofthe present invention are prodrugs which are hydrolyzed to camptothecinanalogs demonstrating inhibitory activity on topoisomerase I. Thecamptothecin analogs formed by hydrolysis of the esters of the inventionare also effective in treating leukemia and solid tumors in mammals.Numerous camptothecin analogs have been shown to be effective againstleukemia using the standard L1210 leukemia assay (Wall et al. (1993),Journal of Medicinal Chemistry, 36: 2689-2700). High activity ofcamptothecin and camptothecin analogs has also been shown in the P388leukemia assay (Wall (1983), Medical and Pediatric Oncology, 11:480A-489A). The later reference also provides a correlation between antileukemia activity as determined by the L1210 and the P388 leukemiaassays with efficacy of camptothecin analogs against solid tumors.Compounds reported as active in the leukemia assays also havedemonstrated activity in a number of solid tumors including a colonxenograft, a lung xenograft, a Walker sarcoma and a breast xenograft(Wall (1983), Table IV, page 484 A). Recent studies have confirmed thecorrelation between topoisomerase I inhibitory activity and antileukemia/anti tumor activity of camptothecin analogs (Giovanella et al.(1989), Science, 246: 1046-1048). The compounds of the present inventionare particularly effective in the treatment of colon, lung, breast andovary solid tumors, brain glioma and leukemia. These compounds may alsobe used to treat malaria.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A camptothecin analog having the structure:

where X and Y are each independently NO₂, NH₂, H. F. Cl, Br, I, COOH, OH, O-C₁₋₆ alkyl, SH, S—C₁₋₆ alkyl, CN, NH—C₁₋₆ alkyl, N(C₁₋₆ alkyl)₂, CHO, C₁₋₈ alkyl, N₃, -Z-(CH₂)_(a)-N-((CH₂)_(b)OH)₂, wherein Z is selected from the group consisting of O, NH and S, and a and b are each independently an integer of 2 or 3, -Z-(CH₂)_(a)—N—(C₁₋₆ alkyl)₂ wherein Z is selected from the group consisting of O, NH and S, and a is an integer of 2 or 3, or —CH₂-L, where L is halogen (F, Cl, Br, I), ⁺N₂, ⁺(OR¹)₂, ⁺S(R¹)₂, ⁺N(R¹)₃, OC(O)R¹, OSO₂R¹, OSO₂CF₃, OSO₂C₄F₉, C₁₋₆ alkyl-C(═O)—, C₄₋₁₈ aryl-C(═O)—, C₁₋₆ alkyl-SO₂—, perfluoro C₁₋₆ alkyl-SO₂— or C₄₋₁₈ aryl-SO₂—, (where each R¹ independently is C₁₋₆ alkyl, C₄₋₁₈ aryl or C₄₋₁₈ ArC₁₋₁₆ alkyl); or —CH₂NR²R³, where (a) R² and R³ are, independently, hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl C₁₋₆ alkyl, C₂₋₆ alkenyl, hydroxy C₁₋₆ alkyl, C₁₋₆ alkoxy C₁₋₆ COR⁴ where R⁴ is hydrogen, C₁₋₆ alkyl, perhalo C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl, hydroxyl-C₁₋₆ alkyl, C₁₋₆-alkoxy, or C₁₋₆ alkoxy-C₁₋₆ alkyl, or (b) R² and R³ taken together with the nitrogen atom to which they are attached form a saturated 3-7 membered heterocyclic ring which may contain a O, S or NR⁵ group, where R⁵ is hydrogen, C₁₋₆ alkyl, perhalo-C₁₋₆ alkyl, aryl, aryl substituted with one or more groups selected from the group consisting of C₁₋₆ alkyl, halogen, nitro, amino, C₁₋₆ alkylamino, perhalo-C₁₋₆ alkyl, hydroxyl-C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl and —COR⁶ where R⁶ is hydrogen, C₁₋₆ alkyl perhalo-C₁₋₆ alkyl, C₁₋₆ alkoxy, aryl, and aryl substituted with one or more C₁₋₆ alkyl, perhalo-C₁₋₆ alkyl, hydroxyl-C₁₋₆ alkyl, or C₁₋₆ alkoxy-C₁₋₆ alkyl groups; R⁷ is C(O)—(CH₂)_(m)—NR⁸R⁹, where m is an integer of 1-6 or —C(O)CHR¹⁰NR⁸R⁹, where R¹⁰ is the side chain of one of the naturally occurring α-amino acids, R⁸ and R⁹ are, independently, hydrogen, C₁₋₈ alkyl or —C(O)CHR¹¹NR¹²R¹²′ where R¹¹ is the side chain of one of the naturally occurring α-amino acids and R¹² and R¹²′ are each independently hydrogen or C₁₋₈ alkyl; W is independently H or F, R¹³ and R¹⁴ are each H or combine to form a double bond; and n is an integer of 1 or 2, and salts thereof.
 2. The camptothecin analog of claim 1, wherein n is
 1. 3. The camptothecin analog of claim 1, wherein Y is —CH₂-L.
 4. The camptothecin analog of claim 1, wherein L is selected from the group consisting of Cl, Br and I.
 5. (canceled)
 6. The camptothecin analog of claim 1, which is selected from the group consisting of R isomers, S isomers and mixtures thereof.
 7. The camptothecin analog of claim 6, wherein the analog is the S isomer.
 8. The camptothecin analog of claim 6, wherein tile analog is the R isomer.
 9. The camptothecin analog of claim 6, wherein the analog is an S rich mixture of S and R isomers.
 10. The camptothecin analog of claim 6, wherein the analog is a R rich mixture of S and R isomers.
 11. The camptothecin analog of claim 6, wherein the analog is a racemic mixture of R and S isomers.
 12. A method of treating leukemia or solid tumors comprising administering to a patient in need thereof, the camptothecin analog of claim
 1. 13. A pharmaceutical composition comprising the camptothecin analog of claim
 1. 14. A method for inhibiting the enzyme topoisomerase I, comprising contacting a DNA-topoisomerase I complex with the camptothecin analog of claim
 1. 15. (canceled) 